Recombinase Switch With GFP (weaker RBS)
Figure 1: Diagram showing the construct used for the in vitro characterisation of the switch. Diagram shows the integrase switch containing a HinDIII restriction site. Once exposed to integrase, the inserted DNA sequence results in the promoter (shown by a red arrow) to be orientated towards GFP, resulting in it’s expression. Also seen here is the HinDIII site contained within the switch, which moves when the DNA is inverted.
PhiC31-based recombinase switch with GFP to the right and a leftward facing promoter. PhiC31 integrase switches on GFP expression. GFP contains a weaker ribosome binding site than in BBa_K1463001.
Recombination was tested in vitro by combining plasmid DNA containing the part with purified phiC31 integrase, which causes the DNA sequence to invert. The HindIII sites are placed in such a way that the bands produced by restriction digest, together with PstI, change depending on whether inversion has occurred or not. This pattern can be seen in the gel (Figure 1).
In vitro recombination of BBa_K1463001 and BBa_K1463002 switch using purified phiC31 integrase incubated at 30C for 2 hours. Plasmid DNA was then cut with PstI and HindIII, which produces different fragment sizes depending on the orientation of the switch. This was run on a 1.0% agarose gel. The gel shows a duplication of the same experiment.GFP32 and GFP denote the two different ribosome binding sites - BBa_B0034 and BBa_B0032
1. Switch & GFP34 – 8uM int. 2. Switch & GFP34 – 4uM int. 3. Switch & GFP34 – 0uM int.
4. Switch & GFP32 – 8uM int. 5. Switch & GFP32 – 4uM int. 6. Switch & GFP34 – 0uM int.
7. Switch & GFP34 – 8uM int. 8.Switch & GFP34 – 4uM int. 9. Switch & GFP34 – 0uM int.
10. Switch & GFP32 – 8uM int. 11. Switch & GFP32 – 4uM int. 12. Switch & GFP34 – 0uM int.
After the recombination reaction the promoter is orientated in the correct direction to drive expression of GFP. This can be seen when the plasmid is transformed into cells. Figure 2 shows the difference between colonies expressing GFP and those which are not. Also shown in Figure 2 is the comparison with part BBa_K1463001, where it can be seen the weaker ribosome binding site BBa_B0032 produces less brightly fluorescing colonies.
Transformed colonies containing the switch plus GFP. The weaker ribosome binding site produces less brightly fluorescing colonies. Colonies transformed with plasmids that were never exposed to integrase do not fluoresce.
1. GFP34 + 8uM int. 2. GFP32 + 8uM int. 3. GFP32 + 0uM int.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 82
Illegal NheI site found at 105
- 21Illegal BamHI site found at 62
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI.rc site found at 938